Ultrahydrogel 250

To analyze the lignin via size-exclusion chromatography, the lyophilized lignin samples were dissolved in a 80:20 (v/v) solution of 0.1 M NaNO₃:0.005 M NaOH/CH₃CN, syringe-filtered (0.22 μm), and loaded onto an Agilent 1260 series HPLC equipped with a Waters Ultrahydrogel ™ 250 (Milford, MA, USA) column. The mobile phase was also an 80:20 (v/v) mixture of 0.1 M NaNO₃:0.005 M NaOH/CH₃CN at a flow rate of 0.6 mL min⁻¹ at 45 °C. The lignin was detected using an Agilent 1260 infinity refractive index detector (RID) detector. Polyethylene glycol (PEG) standards (0.4, 0.6, 1.0, 1.4, 4.3, 6.7, 12.6, and 20.6 kDa; Waters™) were run on the system to generate a standard curve. Based on the reference elution volumes, the number average (M_n) and weight average (M_w) molecular weights were calculated. The number of monomeric units was calculated by utilizing an approximate molecular weight of a lignin monomer as ~187 g/mol. 2D HSQC NMR was performed on the solubilized lignin according to the procedure outlined in Li et al. [58 (link)].

The purity and molecular weight of RATAPW were analyzed by high-performance gel permeation chromatography (HPGPC) performed on three columns (Waters Ultrahydrogel 250, 1,000, and 2,000; 30 cm × 7.8 mm; 6 μm particles) in series (12 (link)). The purified RATAPW was eluted with 3 mmol/L sodium acetate at 0.5 ml/min. Notably, 5.2, 11.6, 23.8, 48.6, 148, 273, and 410 kDa dextrans were used as standards. The calibration curve is calculated using Log (Mw) = –0.1719T + 11.585 (T: elution time).

High-performance size-exclusion chromatography (HPSEC) (HPLC unit, Agilent technologies, Diegem, Belgium) equipped with multi-angle laser light scattering (MALLS) (PN3621, Postnova analytics, Landsberg, Germany) and refractive index (RI) detection (Shodex RI-101, Showa Denko K.K., Kawazaki, Japan) was used to measure the molar mass distribution of the pectin samples following the procedure described by Shpigelman et al. [44 (link)], with minor modifications. Briefly, pectin samples (0.2% w/v) were dissolved overnight in a 0.1 M acetic acetate buffer (pH 4.4) containing 0.1 M NaNO_3. The pectin samples were subsequently filtered (Chromafil^® A-45/25, 0.45 mm pore size, Macherey-Nagel Gmbh, Duren, Germany), injected (100 μL), and separated using a series of Waters columns: Ultrahydrogel 250, 1000, and 2000, with exclusion limits of 8 × 10⁴, 4 × 10⁶, and 1 × 10⁷ g/mol, respectively (Waters, Milford, MA, USA). Elution of the pectin polymers was accomplished with a 0.1 M acetate buffer (pH 4.4) containing 0.1 M NaNO₃ at 35 °C and a flow rate of 0.5 mL/min. The dn/dc value used to calculate the concentration was 0.146 mL/g. Analysis of the samples was performed in duplicate. Calculation of the average molecular weight was achieved by applying the Debye fitting method (2nd order) of the operating software (Nova Mals, version 1.0.0.18, Postnova analytics, Lansberg, Germany).

GPC analysis was performed with a high performance liquid chromatography system equipped with a Shimadzu refractive index detector (Model RID-10A), and a Shimadzu LC-20AT pump. Polysaccharides were analyzed using two columns connected in series: a Supelco Progel-TKS G4000 and a Waters Ultrahydrogel 250 (300 × 7.8 mm). A degassed solution of 0.05 M NaNO₃ prepared with ultra-pure water containing 0.02% NaN₃ was used as solvent and eluent. One hundred μl of polysaccharide solutions (5 mg/ml) were filtered through a 0.22 μm PVDF membrane (GV, Millipore) and injected into the column using a manual valve. The eluent flow rate was of 0.6 ml/min and the temperature was held at 25 °C. The column was calibrated using dextran standards of different molecular weights (Sigma Aldrich, USA).

We analyzed the homogeneity and molecular weight of RGP-1a and RGP-1b using high-performance gel permeation chromatography (HPGPC). The Waters 600 HPLC System (Waters corporation, Milford, MA, USA) equipped with a 2414 differential refractive index detector (RID) and three columns in series (Waters Ultrahydrogel 250, 1000 and 2000; 30 cm × 7.8 mm; 6 μm particles) [34 (link)]. The columns were calibrated using T-series dextrans (molecular weights 5.2, 10, 48.6, 668 and 2000 kDa) and eluted with sodium acetate (3 mM) at a flow rate of 0.5 mL/min. The log (molecular weight) versus elution time (t) calibration curve was based on the following equation:

The molecular weight of BBWPW was estimated by high-performance gel permeation chromatography (HPGPC). Three high-performance gel columns (Waters Ultrahydrogel 250, 1000 and 2000) with the same size (30 cm × 7.8 mm; 6 µm particles) were connected in series. The standard Dextrans (5.2, 23.8, 48.6, 148, 410 kDa) and BBWPW were eluted at 0.5 mL/min with 3 mM sodium acetate. The standard curve of log (Mw) vs. elution time (T) is: log (Mw) = −0.1719T + 11.58. In addition, the carbohydrate content of BBWPW was measured by the phenol–sulphuric acid method [19 (link)].